Jung San Huang

Interactions of High Affinity Insulin-like Growth Factor-binding Proteins with the Type V Transforming Growth Factor-β Receptor in Mink Lung Epithelial Cells

High affinity insulin-like growth factor-binding proteins (IGFBP-1 to -6) are a family of structurally homologous proteins that induce cellular responses by insulin-like growth factor (IGF)-dependent and -independent mechanisms. The IGFBP-3 receptor, which mediates the IGF-independent growth inhibitory response, has recently been identified as the type V transforming growth factor-β receptor (TβR-V) (Leal, S. M., Liu, Q. L., Huang, S. S., and Huang, J. S. (1997) J. Biol. Chem. 272, 20572–20576). To characterize the interactions of high affinity IGFBPs with TβR-V, mink lung epithelial cells (Mv1Lu cells) were incubated with 125I-labeled recombinant human IGFBPs (125I-IGFBP-1 to -6) in the presence of the cross-linking agent disuccinimidyl suberate and analyzed by 5% SDS-polyacrylamide gel electrophoresis and autoradiography.125I-IGFBP-3, -4, and -5 but not 125I-IGFBP-1, -2, and -6 bound to TβR-V as demonstrated by the detection of the ∼400-kDa 125I-IGFBP·TβR-V cross-linked complex in the cell lysates and immunoprecipitates. The analyses of125I-labeled ligand binding competition and DNA synthesis inhibition revealed that IGFBP-3 was a more potent ligand for TβR-V than IGFBP-4 or -5. Most of the high affinity 125I-IGFBPs formed dimers at the cell surface. The cell-surface dimer of125I-IGFBP-3 preferentially bound to and was cross-linked to TβR-V in the presence of disuccinimidyl suberate. IGFBP-3 did not stimulate the cellular phosphorylation of Smad2 and Smad3, key transducers of the transforming growth factor-β type I/type II receptor (TβR-I·TβR-II) heterocomplex-mediated signaling. These results suggest that IGFBP-3, -4, and -5 are specific ligands for TβR-V, which mediates the growth inhibitory response through a signaling pathway(s) distinct from that mediated by the TβR-I and TβR-II heterocomplex.

Synthetic TGF-β antagonist accelerates wound healing and reduces scarring

Wound healing consists of re‐epithelialization, contraction and formation of granulation and scar tissue. TGF‐β is involved in these events, but its exact roles are not well understood. Here we demonstrate that topical application of a synthetic TGF‐β antagonist accelerates re‐epithelialization in pig burn wounds (100% re‐epithelialization in antagonist‐treated wounds vs. ~ 70% reepithelialization in control wounds on postburn day 26) and reduces wound contraction and scarring in standard pig skin burn, pig skin excision and rabbit skin excision wounds. These results support the distinct roles of TGF‐β in the complex process of wound healing and demonstrate the feasibility of manipulating wound healing by TGF‐β antagonist.

Cholesterol modulates cellular TGF‐β responsiveness by altering TGF‐β binding to TGF‐β receptors

Transforming growth factor‐β (TGF‐β) responsiveness in cultured cells can be modulated by TGF‐β partitioning between lipid raft/caveolae‐ and clathrin‐mediated endocytosis pathways. The TβR‐II/TβR‐I binding ratio of TGF‐β on the cell surface has recently been found to be a signal that controls TGF‐β partitioning between these pathways. Since cholesterol is a structural component in lipid rafts/caveolae, we have studied the effects of cholesterol on TGF‐β binding to TGF‐β receptors and TGF‐β responsiveness in cultured cells and in animals. Here we demonstrate that treatment with cholesterol, alone or complexed in lipoproteins, decreases the TβR‐II/TβR‐I binding ratio of TGF‐β while treatment with cholesterol‐lowering or cholesterol‐depleting agents increases the TβR‐II/TβR‐I binding ratio of TGF‐β in all cell types studied. Among cholesterol derivatives and analogs examined, cholesterol is the most potent agent for decreasing the TβR‐II/TβR‐I binding ratio of TGF‐β. Cholesterol treatment increases accumulation of the TGF‐β receptors in lipid rafts/caveolae as determined by sucrose density gradient ultracentrifugation analysis of cell lysates. Cholesterol/LDL suppresses TGF‐β responsiveness and statins/β‐CD enhances it, as measured by the levels of P‐Smad2 and PAI‐1 expression in cells stimulated with TGF‐β. Furthermore, the cholesterol effects observed in cultured cells are also found in the aortic endothelium of atherosclerotic ApoE‐null mice fed a high cholesterol diet. These results indicate that high plasma cholesterol levels may contribute to the pathogenesis of certain diseases (e.g., atherosclerosis) by suppressing TGF‐β responsiveness. J. Cell. Physiol. 215: 223–233, 2008.

Cellular Heparan Sulfate Negatively Modulates Transforming Growth Factor-β1 (TGF-β1) Responsiveness in Epithelial Cells

Cell-surface proteoglycans have been shown to modulate transforming growth factor (TGF)-β responsiveness in epithelial cells and other cell types. However, the proteoglycan (heparan sulfate or chondroitin sulfate) involved in modulation of TGF-β responsiveness and the mechanism by which it modulates TGF-β responsiveness remain unknown. Here we demonstrate that TGF-β1 induces transcriptional activation of plasminogen activator inhibitor-1 (PAI-1) and growth inhibition more potently in CHO cell mutants deficient in heparan sulfate (CHO-677 cells) than in wild-type CHO-K1 cells. 125I-TGF-β1 affinity labeling analysis of cell-surface TGF-β receptors reveals that CHO-K1 and CHO-677 cells exhibit low (<1) and high (>1) ratios of 125I-TGF-β1 binding to TβR-II and TβR-I, respectively. Receptor-bound 125I-TGF-β1 undergoes nystatin-inhibitable rapid degradation in CHO-K1 cells but not in CHO-677 cells. In Mv1Lu cells (which, like CHO-K1 cells, exhibit nystatin-inhibitable rapid degradation of receptor-bound 125I-TGF-β1), treatment with heparitinase or a heparan sulfate biosynthesis inhibitor results in a change from a low (<1) to a high (>1) ratio of 125I-TGF-β1 binding to TβR-II and TβR-I and enhanced TGF-β1-induced transcriptional activation of PAI-1. Sucrose density gradient analysis indicates that a significant fraction of TβR-I and TβR-II is localized in caveolae/lipid-raft fractions in CHO-K1 and Mv1Lu cells whereas the majority of the TGF-β receptors are localized in non-lipid-raft fractions in CHO-677 cells. These results suggest that heparan sulfate negatively modulates TGF-β1 responsiveness by decreasing the ratio of TGF-β1 binding to TβR-II and TβR-I, facilitating caveolae/lipid-raft-mediated endocytosis and rapid degradation of TGF-β1, thus diminishing non-lipid-raft-mediated endocytosis and signaling of TGF-β1 in these epithelial cells.

TRANSFORMING GROWTH FACTOR BETA PEPTIDE ANTAGONISTS AND THEIR CONVERSION TO PARTIAL AGONISTS

Transforming growth factor β (TGF-β) has been implicated in the pathogenesis of various human diseases. Synthetic TGF-β antagonists therefore could have therapeutic utility. Here we show the development of such compounds. Three synthetic pentacosapeptides designated β1 25-(41–65), β2 25-(41–65), and β3 25-(41–65), whose amino acid sequences correspond to the 41st to 65th amino acid residues of TGF-β1, TGF-β2, and TGF-β3, respectively, inhibit the binding of 125I-labeled TGF-β isoforms to TGF-β receptors in mink lung epithelial cells with IC50 of ∼0.06–2 μm. β1 25-(41–65) blocks TGF-β1-induced growth inhibition and TGF-β1-induced plasminogen activator inhibitor-1 expression in these cells. The variants designated β1 25-(41–65)W52A/D55A and β3 25-(41–65)R52A/D55A, in which both Trp52/Arg52 and Asp55are replaced by alanine residues, do not have TGF-β antagonist activity. Multiple conjugation of β1 25-(41–65) to carrier proteins enhances its antagonist activity but also confers partial agonist activity as measured by DNA synthesis inhibition. These results suggest that the (W/R)XXD motif is important for the activities of these TGF-β peptide antagonists and that this motif may be the active site sequence of TGF-β.

LRP-1/TβR-V mediates TGF-β1-induced growth inhibition in CHO cells

The type V transforming growth factor‐β (TGF‐β) receptor (TβR‐V) is hypothesized to be involved in cellular growth inhibition by TGF‐β1. Recently, TβR‐V was found to be identical to low density lipoprotein receptor‐related protein‐1 (LRP‐1). Here we demonstrate that TGF‐β1 inhibits growth of wild‐type CHO cells but not LRP‐1‐deficient mutant cells (CHO‐LRP‐1− cells). Stable transfection of CHO‐LRP‐1− cells with LRP‐1 cDNA restores the wild‐type morphology and the sensitivity to growth inhibition by TGF‐β1. In addition, overexpression of LRP‐1 minireceptors exerts a dominant negative effect and attenuates the growth inhibitory response to TGF‐β1 in wild‐type CHO cells. These results suggest that LRP‐1/TβR‐V is critical for TGF‐β1‐mediated growth inhibition in CHO cells.

FUNCTION OF THE TYPE V TRANSFORMING GROWTH FACTOR BETA RECEPTOR IN TRANSFORMING GROWTH FACTOR BETA -INDUCED GROWTH INHIBITION OF MINK LUNG EPITHELIAL CELLS

The type V transforming growth factor β (TGF-β) is a 400-kDa nonproteoglycan membrane protein that co-expresses with the type I, type II, and type III TGF-β receptors in most cell types. The type V TGF-β receptor exhibits a Ser/Thr-specific protein kinase activity with distinct substrate specificity (Liu, Q., Huang, S. S., and Huang, J. (1994) J. Biol. Chem. 269, 9221–9226). In mink lung epithelial cells, the type V TGF-β receptor was found to form heterocomplexes with the type I TGF-β receptor by immunoprecipitation with antiserum to the type V TGF-β receptor after 125I-TGF-β affinity labeling or Trans35S-label metabolic labeling of the cells. The kinase activity of the type V TGF-β receptor was stimulated after treatment of mink lung epithelial cells with TGF-β. TGF-β stimulation resulted in the growth inhibition of wild-type mink lung epithelial cells and to a lesser extent of the type I and type II TGF-β receptor-defective mutants, although higher concentrations of TGF-β were required for the growth inhibition of these mutants. TGF-β was unable to induce growth inhibition in human colorectal carcinoma cells lacking the type V TGF-β receptor but expressing the type I and type II TGF-β receptors. These results suggest that the type V TGF-β receptor can mediate the TGF-β-induced growth inhibitory response in the absence of the type I or type II TGF-β receptor. These results also support the hypothesis that loss of the type V TGF-β receptor may contribute to the malignancy of certain carcinoma cells.

A mechanism by which dietary trans fats cause atherosclerosis

Dietary trans fats (TFs) have been causally linked to atherosclerosis, but the mechanism by which they cause the disease remains elusive. Suppressed transforming growth factor (TGF)-β responsiveness in aortic endothelium has been shown to play an important role in the pathogenesis of atherosclerosis in animals with hypercholesterolemia. We investigated the effects of a high TF diet on TGF-β responsiveness in aortic endothelium and integration of cholesterol in tissues. Here, we show that normal mice fed a high TF diet for 24 weeks exhibit atherosclerotic lesions and suppressed TGF-β responsiveness in aortic endothelium. The suppressed TGF-β responsiveness is evidenced by markedly reduced expression of TGF-β type I and II receptors and profoundly decreased levels of phosphorylated Smad2, an important TGF-β response indicator, in aortic endothelium. These mice exhibit greatly increased integration of cholesterol into tissue plasma membranes. These results suggest that dietary TFs cause atherosclerosis, at least in part, by suppressing TGF-β responsiveness. This effect is presumably mediated by the increased deposition of cholesterol into cellular plasma membranes in vascular tissue, as in hypercholesterolemia.

Identification of insulin receptor substrate proteins as key molecules for the TβR-V/LRP-1-mediated growth inhibitory signaling cascade in epithelial and myeloid cells

The type V TGF‐β receptor (TβR‐V) mediates IGF‐independent growth inhibition by IGFBP‐3 and mediates growth inhibition by TGF‐β 1 in concert with the other TGF‐β receptor types. TβR‐V was recently found to be identical to LRP‐1. Here we find that insulin and (Q3A4Y15L16) IGF‐I (an IGF‐I analog that has a low affinity for IGFBP‐3) antagonize growth inhibition by IGFBP‐3 in mink lung epithelial cells (Mv1Lu cells) stimulated by serum. In these cells, IGFBP‐3 induces serine‐specific dephosphorylation of IRS‐1 and IRS‐2. The IGFBP‐3‐induced dephosphorylation of IRS‐2 is prevented by cotreatment of cells with insulin, (Q3A4Y15L16) IGF‐I, or TβR‐V/LRP‐1 antagonists. The magnitude of the IRS‐2 dephosphorylation induced by IGFBP‐3 positively correlates with the degree of growth inhibition by IGFBP‐3 in Mv1Lu cells and mutant cells derived from Mv1Lu cells. Stable transfection of murine 32D myeloid cells (which lack endogenous IRS proteins and are insensitive to growth inhibition by IGFBP‐3) with IRS‐1 or IRS‐2 cDNA confers sensitivity to growth inhibition by IGFBP‐3; this IRS‐mediated growth inhibition can be completely reversed by insulin in 32D cells stably expressing IRS‐2 and the insulin receptor. These results suggest that IRS‐1 and IRS‐2 are key molecules for the TβR‐V/LRP‐1‐mediated growth inhibitory signaling cascade.

Amyloid β-Peptide Possesses a Transforming Growth Factor-β Activity

Amyloid β-peptide (Aβ) of 39–42 amino acid residues is a major constituent of Alzheimer’s disease neurite plaques. Aβ aggregates (fibrils) are believed to be responsible for neuronal damage and dysfunction, as well as microglia and astrocyte activation in disease lesions by multiple mechanisms. Since Aβ aggregates possess the multiple valencies of anFAED motif (20th to 23rd amino acid residues), which resembles the putative transforming growth factor-β (TGF-β) active site motif, we hypothesize that Aβ monomers and Aβ aggregates may function as TGF-β antagonists and partial agonists, analogous to previously described monovalent and multivalent TGF-β peptide antagonists and agonists (Huang, S. S., Liu, Q., Johnson, F. E., Konish, Y., and Huang, J. S. (1997) J. Biol. Chem. 272, 27155–27159). Here, we report that the Aβ monomer, Aβ-(1–40) and its fragment, containing the motif inhibit radiolabeled TGF-β binding to cell-surface TGF-β receptors in mink lung epithelial cells (Mv1Lu cells). Aβ-(1–40)-bovine serum albumin conjugate (Aβ-(1–40)-BSA), a multivalent synthetic analogue of Aβ aggregates, exhibited cytotoxicity toward bovine cerebral endothelial cells and rat post-mitotic differentiated hippocampal neuronal cells (H19-7 cells) and inhibitory activities of radiolabeled TGF-β binding to TGF-β receptors and TGF-β-induced plasminogen activator inhibitor-1 expression, that were ∼100–670 times more potent than those of Aβ-(1–40) monomers. At less than micromolar concentrations, Aβ-(1–40)-BSA but not Aβ-(1–40) monomers inhibited proliferation of Mv1Lu cells. Since TGF-β is an organizer of responses to neurodegeneration and is also found in neurite plaques, the TGF-β antagonist and partial agonist activities of Aβ monomers and aggregates may play an important role in the pathogenesis of the disease.